Modified vasoactive intestinal peptides

ABSTRACT

The present invention provides modified Vasoactive Intestinal Peptides (VIPs), encoding polynucleotides and vectors, as well as pharmaceutical compositions comprising the same. The invention further provides methods of making and using the modified VIP agents. In accordance with the invention the VIP exhibits an extended circulatory half-life, receptor-binding or biological potency, and/or altered receptor binding profile with respect to unmodified VIP.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/681,597, filed Apr. 8, 2015, which is a continuation of U.S.application Ser. No. 12/857,103, filed Aug. 16, 2010, now U.S. Pat. No.9,029,505, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/234,151, filed Aug. 14, 2009, whichis herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to vasoactive intestinal peptides (VIPs)and pharmaceutical compositions comprising the same, including VIPshaving an extended circulatory half-life, and VIPs having receptorbinding profiles that differ from the unmodified mature peptide.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename: PHAS_019_03US_SeqList_ST25.txt, date recorded: Jun. 13, 2017, file size 44kilobytes).

BACKGROUND

Vasoactive intestinal peptide (VIP) has a number of biological effectsincluding with respect to hemostasis, the immune system, and the nervoussystem. See, Delgado et al., The Significance of Vasoactive IntestinalPeptide in Immunomodulation, Pharmacol. Reviews 56(2):249-290 (2004).For example, VIP has a beneficial effect on blood and pulmonary pressureand on a wide range of immunological and inflammatory conditions. VIPhas great potential as an active agent for pulmonary hypertension,chronic obstructive pulmonary disease (COPD), arthritis, inflammatorybowel disease (IBD), and asthma to mention a few.

There are at least two receptors for VIP, including VPAC1 and VPAC2.These receptors bind both VIP and the related molecule pituitaryadenylate cyclase-activating polypeptide (PACAP) to some degree. Bothreceptors are members of the 7-transmembrane G-protein coupled receptorfamily. VPAC1 is distributed, for example, in the CNS, liver, lung,intestine and T-lymphocytes. VPAC2 is found, for example, in the CNS,pancreas, skeletal muscle, heart, kidney, adipose tissue, testis, andstomach.

The short half-life of VIP renders this peptide impractical as apharmaceutical agent. See Pozo D, et al., Tuning immune tolerance withvasoactive intestinal peptide: A new therapeutic approach for immunedisorders. Peptides 28(9):1833-1846 (2007). Indeed, studies have shownthat the half-life of VIP in blood is less than 2 minutes (Domschke etal., 1978, Gut 19: 1049-53; Burhol et al., 1978, Scand J Gastroent 13:807-813). Further, the multitude of biological effects of VIP maycomplicate its development for any particular indication. Modifiedversions of VIP are therefore needed to render the agent therapeuticallypractical, for example, by extending half-life and/or designingmolecules having desirable receptor-binding profiles.

SUMMARY OF THE INVENTION

The present invention provides modified Vasoactive Intestinal Peptides(VIPs), as well as encoding polynucleotides and vectors, andpharmaceutical compositions comprising the modified VIPs. The inventionfurther provides methods of making the modified VIP molecules, andmethods of using the modified VIP agents for the treatment of patients.In accordance with the invention, the modified VIP exhibits an extendedcirculatory half-life or persistence in the body, and/or comparablereceptor-binding and/or biological potency, and/or altered receptorbinding profile with respect to unmodified VIP.

In one aspect, the invention provides modified VIP molecules andpharmaceutical compositions comprising the same. The modified VIPmolecules are recombinantly or chemically modified at the N- and/orC-termini by addition of one or more amino acids, and/or by fusion toheterologous amino acid sequences, so as to provide a longer circulatoryhalf-life or persistence in the body, comparable biological potency,and/or a modified receptor binding profile. For example, in someembodiments, the 28-amino acid mature VIP, which begins with anN-terminal His, comprises additional N-terminal amino acids, such as asingle amino acid at the N-terminus (e.g., Met). In these or otherembodiments, the modified VIP contains an N- or C-terminal fusion to anElastin-Like-Peptide (ELP) as described herein. Such modified VIPmolecules may show an increased circulatory half-life or persistence inthe body, and/or an altered binding preference for VPAC2 over VPAC1.

For example, a VIP may be fused (e.g., by recombinant means) to theN-terminus of an ELP. The histidine of the natural, mature VIP may be atthe N-terminus. Such therapeutic agents may require significantly lessfrequent dosing than the unfused counterpart, such as dosing of fromabout 1-7 times per week (e.g. daily or weekly dosing).

In alternative embodiments, the VIP-ELP fusion may contain a methionineat the N-terminus with the His of the natural mature VIP product atposition 2. In yet other embodiments, the VIP-ELP molecule starts withmethionine alanine alanine. When produced in bacteria, the firstmethionine is lost and the product contains Ala-Ala at the N-terminus.Ala-Ala is removed in vitro or in vivo by the action of DPP-IVpeptidase, thereby leaving the natural mature VIP N-terminus. Theseconstructs containing additional amino acids at the N-terminus exhibit asignificantly different activity when tested for binding activity atVPAC1 and VPAC2 receptors. For example, while both constructs canactivate the VPAC2 receptor with a similar EC50, a construct withmethionine at the N-terminus (and His at position 2) is at least 100fold less active at the VPAC1 receptor.

In various embodiments, the modified VIP of the invention having anN-terminal Met has the advantage of being obtainable by recombinantmeans, such as by production in E. coli or other expression system,without further post-expression manufacturing processes to expose thenatural or desired VIP N-terminus.

In still other embodiments, VIP may be chemically modified, for example,by the addition of one or more PEG or other chemical moieties (e.g., ator near the N-terminus), as described in detail herein.

In other aspects, the present invention provides polynucleotides andvectors encoding the modified VIP of the invention, as well as hostcells containing the same. The host cells may be suitable forrecombinant production of the modified VIP, such as bacterial or yeastcells suitable for use with known expression systems.

In other aspects, the invention provides methods of treating,ameliorating, or preventing a condition in a mammal. Such conditionsinclude a variety of cardiovascular, immunological (e.g., autoimmune),and neurological conditions. For example, the modified VIP may be usedto adjust the balance between pro-inflammatory and anti-inflammatoryeffectors in a patient, including a patient suffering from an autoimmunedisease or inflammatory condition. Exemplary indications for themodified VIP include hypertension, chronic obstructive pulmonary disease(COPD), diabetes, myocardial fibrosis, heart failure, cardiomyopathy,arthritis, inflammatory bowel disease (IBD), and asthma, among others.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence of a modified VIP-ELP fusionprotein (M-VIP-ELP1-120, SEQ ID NO: 14) having Met at the N-terminus and120 ELP1 units (VPGXG, SEQ ID NO: 3) fused to the VIP at the C-terminus.

FIG. 2 shows the amino acid sequence of a modified VIP-ELP fusionprotein (MAA-VIP-ELP1-120, SEQ ID NO: 15) having Met-Ala-Ala at theN-terminus, which is activatable to the natural mature VIP peptide, and120 ELP1 units (VPGXG, SEQ ID NO: 3) fused to the VIP at the C-terminus.

FIG. 3 is a plasmid map of pPB1031, which encodes ELP1-120 forconvenient production of recombinant fusions.

FIG. 4 depicts pPB1046 encoding an M-VIP-ELP1-120 (SEQ ID NOs: 39 and40) fusion protein. Primers (P0045, SEQ ID NO: 31, P0048, SEQ ID NO: 32,and P0065, SEQ ID NO: 34) for construction of the recombinant gene areshown.

FIG. 5 depicts pPB1047 encoding an MAA-VIP-ELP1-120 (SEQ ID NOs: 41 and42) fusion protein. Primers (P0066, SEQ ID NO: 35, P0064, SEQ ID NO: 33,P0067, SEQ ID NO: 36) for construction of the recombinant gene areshown.

FIG. 6 depicts pPB1048 encoding an ELP1-120-VIP (SEQ ID NOs: 43 and 44)fusion protein. Primers for constructing the recombinant gene (P0068,SEQ ID NO: 37, P0069, SEQ ID NO: 38) are shown.

FIG. 7 is a 10% Tris-Acetate NuPAGE gel analysis of purified VIP-ELPfusion proteins, with or without heat denature.

FIG. 8 shows the in vitro activity of native VIP and VIP-ELP fusionproteins PB1046 and PB1047 for VPAC2 receptor.

FIG. 9 shows the in vitro activity of native VIP and VIP-ELP fusionproteins PB1046 and PB1047 for VPAC1 receptor.

FIG. 10 shows the in vivo effect of the VIP-ELP fusion protein on ratblood pressure. Left panel shows systolic blood pressure. Right panelshows diastolic blood pressure. VIP-ELP lowers blood pressure for over a12 hour period.

FIG. 11 is a plasmid map of pPB1120 (SEQ ID NO: 48), which encodesVIP-ELP1-120.

FIG. 12 shows the in vitro activity of native VIP and VIP-ELP fusionproteins PB1120 and PB1046 for VPAC1 receptor.

FIG. 13 shows the in vitro activity of native VIP and VIP-ELP fusionproteins PB1120 and PB1046 for VPAC2 receptor.

FIG. 14A shows the pharmacokinetic profile of the VIP-ELP fusion proteinPB1120 in monkeys (n=3) following single subcutaneous injection of 3mg/kg with linear axes. FIG. 14B shows the pharmacokinetic profile ofthe VIP-ELP fusion protein PB1120 with semi-logarithmic axes.

FIGS. 15A, 15B, and 15C show the average change in systolic, diastolic,and mean arterial pressure, respectively over 3 hr intervals in ratsinjected subcutaneously with PB1120 at 0.1 mg/kg, 1 mg/kg, or 5 mg/kgdosages. FIG. 15D shows the average heart rate of the subject rats over3 hr intervals following administration of PB1120.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides modified Vasoactive Intestinal Peptides(VIPs), encoding polynucleotides and vectors, as well as pharmaceuticalcompositions comprising the modified VIPs. The invention furtherprovides methods of making and using the modified VIP agents for thetreatment of patients. In accordance with the invention the VIP mayexhibit an extended circulatory half-life or persistence in the body,comparable receptor-binding or biological potency, and/or alteredreceptor binding profile with respect to unmodified VIP. In variousembodiments, the compounds of the invention exhibit a reduced dosingfrequency as compared to unmodified counterparts.

Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) is a peptide hormone containing 28amino acid residues and is produced in many areas of the human bodyincluding the gut, pancreas and suprachiasmatic nuclei of thehypothalamus in the brain. VIP exhibits a wide variety of biologicalactions including systemic vasodilation, hypotension, increased cardiacoutput, respiratory stimulation, hyperglycemia, coronary dilation,bronchodilation in animals and humans. VIP also affects the balance ofthe immune system.

VIP has an effect on several parts of the body. With respect to thedigestive system, VIP may induce smooth muscle relaxation (loweresophageal sphincter, stomach, gallbladder), stimulate secretion ofwater into pancreatic juice and bile, and cause inhibition of gastricacid secretion and absorption from the intestinal lumen. Its role in theintestine is to stimulate secretion of water and electrolytes, as wellas dilating intestinal smooth muscle, dilating peripheral blood vessels,stimulating pancreatic bicarbonate secretion, and inhibitinggastrin-stimulated gastric acid secretion. These effects work togetherto increase motility. VIP has the function of stimulating pepsinogensecretion by chief cells.

VIP has been found in the heart and has significant effects on thecardiovascular system. It causes coronary vasodilation, as well ashaving a positive inotropic and chronotropic effect.

VIP is an immunomodulating peptide useful for treating inflammation andTH1-type autoimmune disease (See Delgado et al., The Significance ofVasoactive Intestinal Peptide in Immunomodulation, Pharmacol. Reviews56(2):249-290 (2004)). VIP is useful for the treatment ofneurodegenerative diseases (see U.S. Pat. No. 5,972,883, which is herebyincorporated by reference in its entirety). VIP and its structurallyrelated peptide pituitary adenylate cyclase-activating polypeptide(PACAP) have important therapeutic effects in chronic inflammatoryrheumatic disease, such as osteoarthritis (OA) and rheumatoid arthritis(RA) by down-regulating both the inflammatory and autoimmune componentsof the disease (Juarranz et al., Vasoactive intestinal peptide modulatesproinflammatory mediator synthesis in osteoarthritic and rheumatoidsynovial cells, Rheumatology, 2004, 43:416-422). In addition, VIPparticipates in maintaining immune tolerance by regulating the balancebetween proinflammatory and anti-inflammatory effectors, or by inducingthe emergence of T-cells having a suppressor activity againstauto-reactive T-cells (Pozo et al., Tuning immune tolerance withvasoactive intestinal peptide: A new therapeutic approach for immunedisorders, Peptide, 2007, 28(9):1833-1846).

Mature VIP has 28 amino acid residues with the following sequence:HSDAVFTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 13). VIP results fromprocessing of the 170-amino acid precursor molecule prepro-VIP.Structures of VIP and exemplary analogs have been described in U.S. Pat.Nos. 4,835,252, 4,939,224, 5,141,924, 4,734,400, 4,605,641, 6,080,837,6,316,593, 5,677,419, 5,972,883, 6,489,297, 7,094,755, and 6,608,174,each of which is hereby incorporated by reference in its entirety forall purposes.

A number of mutations to improve peptide stability against proteasesetc. are detailed in the literature (see Onune et al Physicochemical andpharmacological characterization of novel vasoactive intestinal peptidederivatives with improved stability, Eur. J. Pharm. Biopharm. 2009,which is hereby incorporated by reference in its entirety for allpurposes). These modified VIP peptides have sequences of SEQ ID NO. 21(M17L, to prevent oxidation of Met), SEQ ID NO. 22 (K15R, K20R and K21R,to increase proteolytic stability), and SEQ ID NO. 23 (N24A and S25A, toincrease proteolytic/thermal stability). The present invention providesmodified VIP peptides that include one or more of these modifications,and with additional VIP modifications described herein. Examples ofmodified VIP molecules include the modified VIP peptides of SEQ ID NOs.14-15, 17-27, 40, 42, 44, and 50.

In various embodiments described herein, a modified VIP (e.g.,comprising SEQ ID NO: 13) (or a functional analog as described herein)is provided. Generally, functional analogs of VIP, include functionalfragments truncated at the N- or C-terminus by from 1 to 10 amino acids,including by 1, 2, 3, or up to about 5 amino acids (with respect to SEQID NO: 13). Such functional analogs may contain from 1 to 5 amino acidinsertions, deletions, and/or substitutions (collectively) with respectto the native sequence (e.g., SEQ ID NO: 13), and in each case retainingthe activity of the peptide (e.g., through VPAC2 and/or VPAC1 binding).Such activity may be confirmed or assayed using any available assay,including an assay described herein, and including any suitable assay todetermine or quantify an activity described in Delgado et al., TheSignificance of Vasoactive Intestinal Peptide in Immunomodulation,Pharmacol. Reviews 56(2):249-290 (2004). In these or other embodiments,the VIP component of the modified VIP of the invention has at leastabout 50%, 75%, 80%, 85%, 90%, 95%, or 97% identity with the nativemature sequence (SEQ ID NO: 13). The determination of sequence identitybetween two sequences (e.g., between a native sequence and a functionalanalog) can be accomplished using any alignment tool, including Tatusovaet al. Blast 2 sequences-a new tool for comparing protein and nucleotidesequences, FEMS Microbiol Lett. 174:247-250 (1999).

In one aspect, the present invention provides a modified VIP moleculehaving receptor preference for VPAC2 or VPAC1, as compared to unmodifiedVIP (e.g., a peptide consisting of the amino acid sequence of SEQ ID NO:13). For example, the modified VIP may have a relative bindingpreference for VPAC2 over VPAC1 of at least about 2:1, about 5:1, about10:1, about 25:1, about 50:1, about 100:1, about 500:1 or more. In otherembodiments, the modified VIP may have a relative binding preference forVPAC1 over VPAC2 of at least about 2:1, about 5:1, about 10:1, about25:1, about 50:1, about 100:1, about 500:1, or more. For example, incertain embodiments, the modified VIP activates the VPAC2 receptorsubstantially as mature, unmodified, human VIP, that is, with an EC50within a factor of about 2 of mature, unmodified, human VIP (SEQ ID NO:13). However, this same modified VIP is 50- or 100-fold or more lesseffective than mature, unmodified, human VIP in activating the VPAC1receptor.

Such modified VIP molecules may contain modified N-terminal regions,such as an addition of from 1 to about 500 amino acids to the N-terminalhistidine of VIP, which may include heterologous mammalian amino acidsequence. For example, the modified VIP may contain a single methionineat the N-terminal side of the natural N-terminal histidine of matureVIP. This molecule is also conveniently prepared in E. coli or otherbacterial expression system, since the methionine will not be removed byE coli when the adjacent amino acid is histidine. Alternatively, theN-terminal amino acid may be any of the naturally-occurring amino acids,namely alanine, arginine, asparagine, aspartic acid, cysteine, glutamicacid, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, serine, threonine, tryptophan, tyrosine,valine, and proline.

The additional sequence added to the N-terminus of VIP may be of anysequence, including biologically active and biologically inert sequencesof from 1 to about 100, 1 to about 50, 1 to about 20, 1 to about 10, and1 to about 5 amino acids.

The N-terminus of the modified VIP may have the structure M-N, where Mis methionine, and N is the N-terminus of the VIP molecule (e.g., SEQ IDNo. 14, FIG. 1). This methionine supports translation of the protein ina bacterial or eukaryotic host cell. Thus, the modified VIP can be madein a biological system, including bacterial and yeast expression systems(e.g., E. coli). While methionine can sometimes be removed by methionineaminopeptidase (MA) in bacterial expression systems, histidine (H) isone of the least favored residues at position 2 for MA.

In still other embodiments, the N-terminus is modified by fusion with amammalian heterologous protein, such as a mammalian protein effectivefor extending half-life of therapeutic molecules. Such sequences may bemammalian sequences, such as albumin, transferrin, or antibody Fcsequences. Such sequences are described in See U.S. Pat. No. 7,238,667(particularly with respect to albumin conjugates), U.S. Pat. No.7,176,278 (particularly with respect to transferrin conjugates), andU.S. Pat. No. 5,766,883, which are each hereby incorporated by referencein their entireties.

In other embodiments, the VIP is activatable by a peptidase or protease,such as an endogenous peptidase or protease. Such activatable sequencesare described in International Application No. PCT/US2009/068656, whichis hereby incorporated by reference in its entirety. As used herein, theterms “peptidase” and “protease” are interchangeable. For example, theVIP may be designed to be activatable by a dipeptidyl peptidase.Exemplary dipeptidyl peptidases include dipeptidyl peptidase-1 (DPP-I),dipeptidyl peptidase-3 (DPP-III), dipeptidyl peptidase-4 (DPP-IV),dipeptidyl peptidase-6 (DPP-VI), dipeptidyl peptidase-7 (DPP-VII),dipeptidyl peptidase-8 (DPP-VIII), dipeptidyl peptidase-9 (DPP-IX),dipeptidyl peptidase-10 (DPP-X). Substrate sequences for suchdipeptidases are known.

The N-terminus of an activatable VIP may have the structure Z-N, where Zis a substrate for a dipeptidase (e.g., Z is removed by dipeptidaseexposure), and N is the N-terminus of VIP. The activatable VIP may havean N-terminal sequence with the formula M-X-N where M is methionine, Xis Pro, Ala, or Ser, and N is the N-terminal of VIP or VIP analog. Inthis manner, M and X will be sensitive to, and removed by a host cell(e.g., E. coli), and/or a dipeptidase (e.g., DPP-IV), subsequently.Alternatively, the N-terminal sequence of the activatable VIP may beX1-X2-N, where X1 is Gly, Ala, Ser, Cys, Thr, Val, or Pro; X2 is Pro,Ala, or Ser; and N is the N-terminal of VIP. X1-X2 is a substrate fordipeptidase (e.g., DPP-IV), and dipeptidase digestion will expose N, thedesired N-terminus of the VIP or the VIP analog (e.g., SEQ ID NO. 15,FIG. 2). In such embodiments, the protein may be produced by expressionof a construct encoding M-X1-X2-N (where M is methionine) in a host cell(e.g., E. coli), since Gly, Ala, Ser, Cys, Thr, Val, or Pro at thesecond position will signal the removal of the Met, thereby leavingX1-X2 on the N-terminus, which can be activated by a dipeptidase (e.g.,DPP-IV) in vivo. Such activatable VIP molecules, which are activated topossess the natural mature VIP N-terminus, do not show receptorpreference.

In another embodiment, the N-terminus of the modified activatable VIPmay have the structure M-Z-N, where M is methionine, Z is a substratefor a dipeptidase (e.g., Z is removed by dipeptidase exposure), and N isa non-His N-terminal of an active VIP (modified VIP). For example, themodified activatable VIP may have an N-terminal sequence with theformula M-X-N where M is methionine; X is Pro, Ala, or Ser; and N is anon-His N-terminal of the active VIP. In this manner, M and X will besensitive to, and removed by a host cell (e.g., E. coli), and/or adipeptidase (e.g., DPP-IV), subsequently. Alternatively, the N-terminalsequence of the activatable VIP may be X1-X2-N, where X1 is Gly, Ala,Ser, Cys, Thr, Val, or Pro; X2 is Pro, Ala, or Ser; and N is a non-HisN-terminal of the active VIP. X1-X2 is a substrate for dipeptidase(e.g., DPP-IV), and dipeptidase digestion will expose N, the desirednon-His N-terminus of the VIP.

Still in another embodiment, the N-terminus of a modified activatableVIP may have the structure M-Z-S-N, where M is methionine; Z is asubstrate for a dipeptidase (e.g., Z is removed by dipeptidaseexposure); N is the N-terminus of mature VIP (His); and S is one or moreamino acids which will be exposed after dipeptidase digestion, and whichprovide a modified VIP as previously described. For example, themodified activatable VIP may have an N-terminal sequence with theformula M-X-S-N where M is methionine, X is Pro, Ala, or Ser; N is theN-terminal of mature VIP; and S is one or more amino acids which will beexposed after dipeptidase digestion, and will provide receptorpreference. Alternatively, the N-terminal sequence of the activatableVIP may be X1-X2-S-N, where X1 is Gly, Ala, Ser, Cys, Thr, Val, or Pro;X2 is Pro, Ala, or Ser; N is a non-His N-terminal of VIP; and S is oneor more amino acids which will be exposed after dipeptidase digestion.X1-X2 is a substrate for dipeptidase (e.g., DPP-IV), and dipeptidasedigestion will expose S.

In these or other embodiments, N-terminal chemical modifications to theVIP N-terminus may provide receptor preference. Chemical modification ofproteins and methods thereof are well known in the art. Non-limitingexemplary chemical modifications are PEGylation, methylglyoxalation,reductive alkylation, performic acid oxidation, succinylation,aminoethylation, and lipidation (Clifton, New Protein Techniques, NewJersey: Humana Press, 1985. ISBX. 0-89603-126-8. Volume. 3 of. Methodsin Molecular Biology). Chemical groups, such as PEGylation, may beattached by modifications of cysteine, methionine, histidine, lysine,arginine, tryptophan, tyrosine, carboxyl groups have been describedpreviously (see Lundblad, Techniques in Protein Modification, CRC Press,1995).

Fusions to Bioelastic Polymers

In some embodiments, the VIP of the invention contains an N-terminaland/or C-terminal bioelastic polymer component. A “bioelastic polymer”may exhibit an inverse temperature transition. Bioelastic polymers areknown and described in, for example, U.S. Pat. No. 5,520,672 to Urry etal. Bioelastic polymers may be polypeptides comprising elastomeric unitsof pentapeptides, tetrapeptides, and/or nonapeptides (e.g. “elastin-likepeptides”). Bioelastic polymers that may be used to carry out thepresent invention are set forth in U.S. Pat. No. 4,474,851, whichdescribes a number of tetrapeptide and pentapeptide repeating units thatcan be used to form a bioelastic polymer. Specific bioelastic polymersare also described in U.S. Pat. Nos. 4,132,746; 4,187,852; 4,500,700;4,589,882; and 4,870,055. Still other examples of bioelastic polymersare set forth in U.S. Pat. No. 6,699,294, U.S. Pat. No. 6,753,311, andU.S. Pat. No. 6,063,061. The structures of such bioelastic polymers arehereby incorporated by reference.

In one embodiment, the bioelastic polymers are polypeptides of thegeneral formula (VPGXG)_(m) where X is any amino acid (e.g., Ala, Leu,Phe) and m is from about 20 to about 2000, or about 50 to about 180. Inexemplary embodiments, m is 60, 90, 120, 150, or 180. The frequency ofthe various amino acids as the fourth amino acid can be changed, as wellas the identity of X.

For example, bioelastic polymers may comprise repeating elastomericunits selected from bioelastic pentapeptides and tetrapeptides, wherethe repeating units comprise amino acid residues selected from the groupconsisting of hydrophobic amino acid and glycine residues and where therepeating units exist in a conformation having a beta-turn of theformula:

wherein R₁-R₅ represent side chains of amino acid residues 1-5, and m is0 when the repeating unit is a tetrapeptide or 1 when the repeating unitis a pentapeptide. Nonapeptide repeating units generally consist ofsequential tetra- and pentapeptides. Hydrophobic amino acid residues areselected from alanine, valine, leucine, isoleucine, proline,phenylalanine, tryptophan, and methionine. In many cases, the firstamino acid residue of the repeating unit is a residue of valine,leucine, isoleucine or phenylalanine; the second amino acid residue is aresidue of proline; the third amino acid residue is a residue ofglycine; and the fourth amino acid residue is glycine or a veryhydrophobic residue such as tryptophan, phenylalanine or tyrosine.Particular examples include the tetrapeptide Val-Pro-Gly-Gly, thetetrapeptide GGVP, the tetrapeptide GGFP, the tetrapeptide GGAP, thepentapeptide is Val-Pro-Gly-Val-Gly, the pentapeptide GVGVP, thepentapeptide GKGVP, the pentapeptide GVGFP, the pentapeptide GFGFP, thepentapeptide GEGVP, the pentapeptide GFGVP, and the pentapeptide GVGIP.See, e.g., U.S. Pat. No. 6,699,294.

In certain exemplary embodiments, the VIP of the invention contains anN-terminal and/or C-terminal ELP component. The ELP component comprisesor consists of structural peptide units or sequences that are relatedto, or derived from, the elastin protein. Such sequences are useful forimproving the properties of therapeutic proteins in one or more ofbioavailability, therapeutically effective dose and/or administrationfrequency, biological action, formulation compatibility, resistance toproteolysis, solubility, half-life or other measure of persistence inthe body subsequent to administration, and/or rate of clearance from thebody. See, for example, WO 2008/030968 which is hereby incorporated byreference in its entirety.

When the ELP is positioned at the C-terminus of VIP, additionalmodifications may be made at the VIP N-terminus, such as the addition ofone or more amino acids, as described above. In alternative embodiments,there are no such modifications at the VIP N-terminus.

The ELP component is constructed from structural units of from three toabout twenty amino acids, or in some embodiments, from four to ten aminoacids, such as five or six amino acids. The length of the individualstructural units, in a particular ELP component, may vary or may beuniform. In certain embodiments, the ELP component is constructed of apolytetra-, polypenta-, polyhexa-, polyhepta-, polyocta, andpolynonapeptide motif of repeating structural units. Exemplarystructural units include units defined by SEQ ID NOs: 1-12 (see below),which may be employed as repeating structural units, includingtandem-repeating units, or may be employed in some combination, tocreate an ELP effective for improving the properties of the therapeuticcomponent. Thus, the ELP component may comprise or consist essentiallyof structural unit(s) selected from SEQ ID NOS: 1-12, as defined below.

The ELP component, comprising such structural units, may be of varyingsizes. For example, the ELP component may comprise or consistessentially of from about 10 to about 500 structural units, or incertain embodiments about 20 to about 200 structural units, or incertain embodiments from about 50 to about 150 structural units, or fromabout 75 to about 130 structural units, including one or a combinationof units defined by SEQ ID NOS: 1-12. The ELP component may compriseabout 120 structural units, such as repeats of structural units definedby SEQ ID NO: 3 (defined below). Thus, the ELP component may have alength of from about 50 to about 2000 amino acid residues, or from about100 to about 600 amino acid residues, or from about 200 to about 500amino acid residues, or from about 200 to about 400 amino acid residues.

In some embodiments, the ELP component, or in some cases the therapeuticagent, has a size of less than about 150 kDa, or less than about 100kDa, or less than about 55 kDa, or less than about 50 kDa, or less thanabout 40 kDa, or less than about 30 or 25 kDa.

In some embodiments, the ELP component in the untransitioned state mayhave an extended, relatively unstructured and non-globular form so as toescape kidney filtration. In such embodiments, the therapeutic agents ofthe invention have a molecular weight of less than the generallyrecognized cut-off for filtration through the kidney, such as less thanabout 60 kD, or in some embodiments less than about 55, 50, 45, 40, 30,or 25 kDa, and nevertheless persist in the body by at least 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 100-fold longer than anuncoupled (e.g., unfused or unconjugated) therapeutic counterpart.

In these or other embodiments, the ELP component does not substantiallyor significantly impact the biological action of the therapeuticpeptide. Thus, the VIP with ELP fusion of the present invention mayexhibit a potency (biological action) that is the same or similar to itsunfused counterpart. The VIP with ELP fusion of the present inventionmay exhibit a potency or level of biological action (e.g., as tested invitro or in vivo) of from 10-100% of that exhibited by the unfusedcounterpart in the same assay. In various embodiments, the (activated)VIP with ELP fusion of the present invention may exhibit a potency orlevel of biological action (e.g., as tested in vitro or in vivo) of atleast 50%, 60%, 75%, 80%, 90%, 95% or more of that exhibited by theunfused counterpart.

In certain embodiments, the ELP component undergoes a reversible inversephase transition. That is, the ELP components are structurallydisordered and highly soluble in water below a transition temperature(Tt), but exhibit a sharp (2-3° C. range) disorder-to-order phasetransition when the temperature is raised above the Tt, leading todesolvation and aggregation of the ELP components. For example, the ELPforms insoluble polymers, when reaching sufficient size, which can bereadily removed and isolated from solution by centrifugation. Such phasetransition is reversible, and isolated insoluble ELPs can be completelyresolubilized in buffer solution when the temperature is returned belowthe Tt of the ELPs. Thus, the therapeutic agents of the invention can,in some embodiments, be separated from other contaminating proteins tohigh purity using inverse transition cycling procedures, e.g., utilizingthe temperature-dependent solubility of the therapeutic agent, or saltaddition to the medium. Successive inverse phase transition cycles canbe used to obtain a high degree of purity. In addition to temperatureand ionic strength, other environmental variables useful for modulatingthe inverse transition of the therapeutic agents include pH, theaddition of inorganic and organic solutes and solvents, side-chainionization or chemical modification, and pressure.

In certain embodiments, the ELP component does not undergo a reversibleinverse phase transition, or does not undergo such a transition at abiologically relevant Tt, and thus the improvements in the biologicaland/or physiological properties of the molecule (as described elsewhereherein), may be entirely or substantially independent of any phasetransition properties. Nevertheless, such phase transition propertiesmay impart additional practical advantages, for example, in relation tothe recovery and purification of such molecules.

In the practice of the present invention, the ELP component functions tostabilize or otherwise improve the VIP component in the therapeuticcomposition. Subsequent to administration of the coupled VIP-ELPconstruct to the patient in need of the VIP therapeutic agent, the VIPcomponent and the ELP remain coupled with one another while the VIP istherapeutically active, e.g., for treatment or prophylaxis of a diseasestate or physiological condition, or other therapeutic intervention.

In certain embodiments, the ELP component(s) may be formed of structuralunits, including but not limited to:

(SEQ ID NO: 1) (a) the tetrapeptide Val-Pro-Gly-Gly, or VPGG;(SEQ ID NO: 2) (b) the tetrapeptide Ile-Pro-Gly-Gly, or IPGG;(SEQ ID NO: 3) (c) the pentapeptide Val-Pro-Gly-X-Gly, orVPGXG, where X is any natural or non-naturalamino acid residue, and where X optionallyvaries among polymeric or oligomeric repeats; (SEQ ID NO: 4)(d) the pentapeptide Ala-Val-Gly-Val-Pro, or AVGVP; (SEQ ID NO: 5)(e) the pentapeptide Ile-Pro-Gly-X-Gly, orIPGXG, where X is any natural or non-natural aminoacid residue, and where X optionally varies amongpolymeric or oligomeric repeats; (SEQ ID NO: 6)(e) the pentapeptide Ile-Pro-Gly-Val-Gly, or IPGVG; (SEQ ID NO: 7)(f) the pentapeptide Leu-Pro-Gly-X-Gly, orLPGXG, where X is any natural or non-natural aminoacid residue, and where X optionally varies amongpolymeric or oligomeric repeats; (SEQ ID NO: 8)(g) the pentapeptide Leu-Pro-Gly-Val-Gly, or LPGVG; (SEQ ID NO: 9)(h) the hexapeptide Val-Ala-Pro-Gly-Val-Gly, or VAPGVG; (SEQ ID NO: 10)(I) the octapeptide Gly-Val-Gly-Val-Pro-Gly-Val- Gly, or GVGVPGVG;(SEQ ID NO: 11) (J) the nonapeptide Val-Pro-Gly-Phe-Gly-Val-Gly-Ala-Gly, or VPGFGVGAG; and (SEQ ID NO: 12)(K) the nonapeptides Val-Pro-Gly-Val-Gly-Val-Pro- Gly-Gly, or VPGVGVPGG.

Such structural units defined by SEQ ID NOS:1-12 may form structuralrepeat units, or may be used in combination to form an ELP component inaccordance with the invention. In some embodiments, the ELP component isformed entirely (or almost entirely) of one or a combination of (e.g.,2, 3 or 4) structural units selected from SEQ ID NOS: 1-12. In otherembodiments, at least 75%, or at least 80%, or at least 90% of the ELPcomponent is formed from one or a combination of structural unitsselected from SEQ ID NOS: 1-12, and which may be present as repeatingunits.

In certain embodiments, the ELP component(s) contain repeat units,including tandem repeating units, of the pentapeptide Val-Pro-Gly-X-Gly(SEQ ID NO: 3), where X is as defined above, and where the percentage ofVal-Pro-Gly-X-Gly (SEQ ID NO: 3) pentapeptide units taken with respectto the entire ELP component (which may comprise structural units otherthan VPGXG (SEQ ID NO: 3)) is greater than about 75%, or greater thanabout 85%, or greater than about 95% of the ELP component. The ELPcomponent may contain motifs having a 5 to 15-unit repeat (e.g. about10-unit or about 12-unit repeat) of the pentapeptide of SEQ ID NO: 3,with the guest residue X varying among at least 2 or at least 3 of thestructural units within each repeat. The guest residues may beindependently selected, such as from the amino acids V, I, L, A, G, andW (and may be selected so as to retain a desired inverse phasetransition property). Exemplary motifs include VPGXG (SEQ ID NO: 3),where the guest residues are V (which may be present in from 40% to 60%of structural units), G (which may be present in 20% to 40% ofstructural units, and A (which may be present in 10% to 30% ofstructural units). The repeat motif itself may be repeated, for example,from about 5 to about 20 times, such as about 8 to 15 times (e.g., about12 times), to create an exemplary ELP component. The ELP component asdescribed in this paragraph may of course be constructed from any one ofthe structural units defined by SEQ ID NOS: 1-12, or a combinationthereof. An exemplary ELP component is shown in FIG. 1 fused to theC-terminus of VIP.

In some embodiments, the ELP units may form a β-turn structure thatprovides an elastin-like property (e.g., inverse phase transition).Exemplary peptide sequences suitable for creating a β-turn structure aredescribed in International Patent Application PCT/US96/05186, which ishereby incorporated by reference in its entirety. For example, thefourth residue (X) in the elastin pentapeptide sequence, VPGXG (SEQ IDNO: 3), can be altered without eliminating the formation of a β-turn.

In certain embodiments, the ELP components include polymeric oroligomeric repeats of the pentapeptide VPGXG (SEQ ID NO: 3), where theguest residue X is any amino acid. X may be a naturally occurring ornon-naturally occurring amino acid. In some embodiments, X is selectedfrom alanine, arginine, asparagine, aspartic acid, cysteine, glutamicacid, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, serine, threonine, tryptophan, tyrosine andvaline. In some embodiments, X is a natural amino acid other thanproline or cysteine.

The guest residue X (e.g., with respect to SEQ ID NO: 3, or other ELPstructural unit) may be a non-classical (non-genetically encoded) aminoacid. Examples of non-classical amino acids include: D-isomers of thecommon amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid,A-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, Cα-methyl aminoacids, Nα-methyl amino acids, and amino acid analogs in general.

Selection of X may be independent in each ELP structural unit (e.g., foreach structural unit defined herein having a guest residue X). Forexample, X may be independently selected for each structural unit as anamino acid having a positively charged side chain, an amino acid havinga negatively charged side chain, or an amino acid having a neutral sidechain, including in some embodiments, a hydrophobic side chain.

In still other embodiments, the ELP component(s) may include polymericor oligomeric repeats of the pentapeptides VPGXG (SEQ ID NO:3), IPGXG(SEQ ID NO:5) or LPGXG (SEQ ID NO:7), or a combination thereof, where Xis as defined above.

In each embodiment, the structural units, or in some cases polymeric oroligomeric repeats, of the ELP sequences may be separated by one or moreamino acid residues that do not eliminate the overall effect of themolecule, that is, in imparting certain improvements to the therapeuticcomponent as described. In certain embodiments, such one or more aminoacids also do not eliminate or substantially affect the phase transitionproperties of the ELP component (relative to the deletion of such one ormore amino acids).

The structure of the resulting ELP components may be described using thenotation ELPk [X_(i)Y_(j)-n], where k designates a particular ELP repeatunit, the bracketed capital letters are single letter amino acid codesand their corresponding subscripts designate the relative ratio of eachguest residue X in the structural units (where applicable), and ndescribes the total length of the ELP in number of the structuralrepeats. For example, ELP1 [V₅A₂G₃-10] designates an ELP componentcontaining 10 repeating units of the pentapeptide VPGXG (SEQ ID NO:3),where X is valine, alanine, and glycine at a relative ratio of 5:2:3;ELP1 [K₁V₂F₁-4] designates an ELP component containing 4 repeating unitsof the pentapeptide VPGXG (SEQ ID NO:3), where X is lysine, valine, andphenylalanine at a relative ratio of 1:2:1; ELP1 [K₁V₇F₁-9] designates apolypeptide containing 9 repeating units of the pentapeptide VPGXG (SEQID NO:3), where X is lysine, valine, and phenylalanine at a relativeratio of 1:7:1; ELP1 [V₁A₈G₇-10] designates an ELP component containing10 repeating units of the pentapeptide VPGXG (SEQ ID NO:3), where X isvaline, alanine, and glycine at a relative ratio of 1:8:7; ELP1 [V-5]designates a polypeptide containing 5 repeating units of thepentapeptide VPGXG (SEQ ID NO:3), where X is exclusively valine; ELP1[V-20] designates a polypeptide containing 20 repeating units of thepentapeptide VPGXG (SEQ ID NO:3), where X is exclusively valine; ELP2[5] designates a polypeptide containing 5 repeating units of thepentapeptide AVGVP (SEQ ID NO:4); ELP3 [V-5] designates a polypeptidecontaining 5 repeating units of the pentapeptide IPGXG (SEQ ID NO:5),where X is exclusively valine; ELP4 [V-5] designates a polypeptidecontaining 5 repeating units of the pentapeptide LPGXG (SEQ ID NO:7),where X is exclusively valine. Such ELP components as described in thisparagraph may be used in connection with the present invention toincrease the therapeutic properties of the therapeutic component.

Further, the Tt is a function of the hydrophobicity of the guestresidue. Thus, by varying the identity of the guest residue(s) and theirmole fraction(s), ELPs can be synthesized that exhibit an inversetransition over a 0-100° C. range. Thus, the Tt at a given ELP lengthmay be decreased by incorporating a larger fraction of hydrophobic guestresidues in the ELP sequence. Examples of suitable hydrophobic guestresidues include valine, leucine, isoleucine, phenylalanine, tryptophanand methionine. Tyrosine, which is moderately hydrophobic, may also beused. Conversely, the Tt may be increased by incorporating residues,such as those selected from the group consisting of: glutamic acid,cysteine, lysine, aspartate, alanine, asparagine, serine, threonine,glycine, arginine, and glutamine; preferably selected from alanine,serine, threonine and glutamic acid.

The ELP component in some embodiments is selected or designed to providea Tt (under physiological conditions) ranging from about 10 to about 80°C., such as from about 35 to about 60° C., or from about 38 to about 45°C. In some embodiments, the Tt is greater than about 40° C. or greaterthan about 42° C., or greater than about 45° C., or greater than about50° C. The transition temperature, in some embodiments, is above thebody temperature of the subject or patient (e.g., >37° C.) therebyremaining soluble in vivo, or in other embodiments, the Tt is below thebody temperature (e.g., <37° C.) to provide alternative advantages, suchas in vivo formation of a drug depot for sustained release of thetherapeutic agent. See, for example, US 2007/0009602, which is herebyincorporated by reference in its entirety.

The Tt of the ELP component can be modified by varying ELP chain length,as the Tt generally increases with decreasing MW. For polypeptideshaving a molecular weight >100,000, the hydrophobicity scale developedby Urry et al. (PCT/US96/05186, which is hereby incorporated byreference in its entirety) provides one means for predicting theapproximate Tt of a specific ELP sequence. However, in some embodiments,ELP component length can be kept relatively small, while maintaining atarget Tt, by incorporating a larger fraction of hydrophobic guestresidues (e.g., amino acid residues having hydrophobic side chains) inthe ELP sequence. For polypeptides having a molecular weight <100,000,the Tt may be predicted or determined by the following quadraticfunction: Tt=M₀+M₁X+M₂X² where X is the MW of the fusion protein, andM₀=116.21; M₁=−1.7499; M₂=0.010349.

While the Tt of the ELP component, and therefore of the ELP componentcoupled to a therapeutic component, is affected by the identity andhydrophobicity of the guest residue, X, additional properties of themolecule may also be affected. Such properties include, but are notlimited to solubility, bioavailability, persistence, half-life, potencyand safety of the molecule.

In the Examples section below, it is seen that the ELP-coupled VIP agentretains a significant amount of the native VIP's biological activity,relative to unfused forms of VIP. Additionally, it is shown that ELPsexhibit long half-lives. Correspondingly, ELPs can be used in accordancewith the invention to substantially increase (e.g. by greater than 10%,20%, 30%, 50%, 100%, 200% or more, in specific embodiments) thehalf-life of VIP, as conjugated with an ELP, in comparison to thehalf-life of the free (unconjugated) form of the therapeutic agent. Themodified VIP having extended circulatory half-life may be administeredfrom 1 to about 10 times per week, such as from 1 to about 5, or 1 toabout 3 times per week. The modified VIP or pharmaceutical compositioncomprising the same may be administered about once daily, or about everyother day, or about every third day, or about once a week (i.e. onceweekly dosing).

Conjugation and Coupling

A recombinantly-produced VIP fusion protein, in accordance with certainembodiments of the invention, includes the fusion component (e.g., ELP)and a VIP or an analog of VIP associated with one another by geneticfusion. For example, the fusion protein may be generated by translationof a polynucleotide encoding VIP or an analog of VIP cloned in-framewith the ELP component.

In certain embodiments, the ELP component and VIP or an analog of VIPcan be fused using a linker peptide of various lengths to providegreater physical separation and allow more spatial mobility between thefused portions, and thus maximize the accessibility of VIP or an analogof VIP, for instance, for binding to its cognate receptor. The linkerpeptide may consist of amino acids that are flexible or more rigid. Forexample, a flexible linker may include amino acids having relativelysmall side chains, and which may be hydrophilic. Without limitation, theflexible linker may comprise glycine and/or serine residues. More rigidlinkers may contain, for example, more sterically hindering amino acidside chains, such as (without limitation) tyrosine or histidine. Thelinker may be less than about 50, 40, 30, 20, 10, or 5 amino acidresidues. The linker can be covalently linked to and between VIP or ananalog of VIP and an ELP component, for example, via recombinant fusion.

The linker or peptide spacer may be protease-cleavable or non-cleavable.By way of example, cleavable peptide spacers include, withoutlimitation, a peptide sequence recognized by proteases (in vitro or invivo) of varying type, such as Tev, thrombin, factor Xa, plasmin (bloodproteases), metalloproteases, cathepsins (e.g., GFLG, SEQ ID NO: 47,etc.), and proteases found in other corporeal compartments. In someembodiments employing cleavable linkers, the fusion protein may beinactive, less active, or less potent as a fusion, which is thenactivated upon cleavage of the spacer in vivo. Alternatively, where thetherapeutic agent is sufficiently active as a fusion, a non-cleavablespacer may be employed. The non-cleavable spacer may be of any suitabletype, including, for example, non-cleavable spacer moieties having theformula [(Gly)n-Ser]m, where n is from 1 to 4, inclusive, and m is from1 to 4, inclusive. Alternatively, a short ELP sequence different thanthe backbone ELP could be employed instead of a linker or spacer, whileaccomplishing the necessary effect.

In still other embodiments, the therapeutic agent is a recombinantfusion having a therapeutic component flanked on each terminus by an ELPcomponent. At least one of said ELP components may be attached via acleavable spacer, such that the therapeutic component is inactive, butactivated in vivo by proteolytic removal of a single ELP component. Theresulting single ELP fusion being active, and having an enhancedhalf-life (or other property described herein) in vivo.

In other embodiments, the present invention provides chemical conjugatesof a VIP or an analog of VIP and the ELP component. The conjugates canbe made by chemically coupling an ELP component to VIP or an analog ofVIP by any number of methods well known in the art (See e.g. Nilsson etal., 2005, Ann Rev Biophys Bio Structure 34: 91-118). In someembodiments, the chemical conjugate can be formed by covalently linkingVIP or an analog of VIP to the ELP component, directly or through ashort or long linker moiety, through one or more functional groups onthe therapeutic proteinacious component, e. g., amine, carboxyl, phenyl,thiol or hydroxyl groups, to form a covalent conjugate. Variousconventional linkers can be used, e. g., diisocyanates,diisothiocyanates, carbodiimides, bis (hydroxysuccinimide) esters,maleimide-hydroxysuccinimide esters, glutaraldehyde and the like.

Non-peptide chemical spacers can additionally be of any suitable type,including for example, by functional linkers described in BioconjugateTechniques, Greg T. Hermanson, published by Academic Press, Inc., 1995,and those specified in the Cross-Linking Reagents Technical Handbook,available from Pierce Biotechnology, Inc. (Rockford, Ill.), thedisclosures of which are hereby incorporated by reference, in theirrespective entireties. Illustrative chemical spacers includehomobifunctional linkers that can attach to amine groups of Lys, as wellas heterobifunctional linkers that can attach to Cys at one terminus,and to Lys at the other terminus.

In certain embodiments, relatively small ELP components (e.g., ELPcomponents of less than about 30 kDa, 25 kDa, 20 kDa, 15 kDa, or 10kDa), that do not transition at room temperature (or human bodytemperature, e.g., Tt >37° C.), are chemically coupled or crosslinked.For example, two relatively small ELP components, having the same ordifferent properties, may be chemically coupled. Such coupling, in someembodiments, may take place in vivo, by the addition of a singlecysteine residue at or around the C-terminus of the ELP. Such ELPcomponents may each be fused to one or more therapeutic components, soas to increase activity or avidity at the target.

Polynucleotides, Vectors, Host Cells, and Methods for Production

In another aspect, the invention provides polynucleotides comprising anucleotide sequence encoding the modified VIP of the invention. Suchpolynucleotides may further comprise, in addition to sequences encodingVIP or VIP analog and fusion sequences, one or more expression controlelements. For example, the polynucleotide may comprise one or morepromoters or transcriptional enhancers, ribosomal binding sites,transcription termination signals, and polyadenylation signals, asexpression control elements. The polynucleotide may be inserted withinany suitable vector, which may be contained within any suitable hostcell for expression, such as E. coli.

Generally, a vector comprising the polynucleotide can be introduced intoa cell for expression of the therapeutic agent. The vector can remainepisomal or become chromosomally integrated, as long as the insertencoding the therapeutic agent can be transcribed. Vectors can beconstructed by standard recombinant DNA technology. Vectors can beplasmids, phages, cosmids, phagemids, viruses, or any other types knownin the art, which are used for replication and expression in prokaryoticor eukaryotic cells. It will be appreciated by one of skill in the artthat a wide variety of components known in the art (such as expressioncontrol elements) may be included in such vectors, including a widevariety of transcription signals, such as promoters and other sequencesthat regulate the binding of RNA polymerase onto the promoter. Anypromoter known to be effective in the cells in which the vector will beexpressed can be used to initiate expression of the therapeutic agent.Suitable promoters may be inducible or constitutive.

In certain embodiments, the modified VIP is expressed from E. coli orother bacterial expression system. E. coli generally will not removeN-terminal methionine during expression, such that the modified VIPmolecule maintains receptor specificity. Other expression systems may beemployed in accordance with the invention, including yeast expressionsystems, mammalian cell expression systems, and baculovirus systems.

The therapeutic protein, when employing ELP fusion sequences, may berecovered by inverse temperature cycling. Specifically, as previouslydescribed, the ELP component undergoes a reversible inverse phasetransition. That is, the ELP components are structurally disordered andhighly soluble in water below a transition temperature (Tt), but exhibita sharp (2-3° C. range) disorder-to-order phase transition when thetemperature is raised above the Tt, leading to desolvation andaggregation of the ELP components. For example, the ELP forms insolublepolymers, when reaching sufficient size, which can be readily removedand isolated from solution by centrifugation. Such phase transition isreversible, and isolated insoluble ELPs can be completely resolubilizedin buffer solution when the temperature is returned below the Tt of theELPs. Thus, the therapeutic agents of the invention can, in someembodiments, be separated from other contaminating proteins to highpurity using inverse transition cycling procedures, e.g., utilizing thetemperature-dependent solubility of the therapeutic agent, or saltaddition to the medium. Successive inverse phase transition cycles canbe used to obtain a high degree of purity. In addition to temperatureand ionic strength, other environmental variables useful for modulatingthe inverse transition of the therapeutic agents include pH, theaddition of inorganic and organic solutes and solvents, side-chainionization or chemical modification, and pressure.

Pharmaceutical Compositions and Methods of Administration

The present invention further provides pharmaceutical compositionscomprising an effective amount of the modified VIP of the invention (asdescribed above) together with a pharmaceutically acceptable carrier,diluent, or excipient. Such pharmaceutical compositions are effectivefor treating or ameliorating, for example, autoimmune or inflammatorydisease, as described herein.

The therapeutic agents of the invention may be administered per se aswell as in various forms including pharmaceutically acceptable esters,salts, and other physiologically functional derivatives thereof. In suchpharmaceutical formulations, the therapeutic agents can be used solely,or together (including formulated with) other therapeutic ingredients,such as anti-inflammatory agents or immunosuppressants.

The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and not undulydeleterious to the recipient thereof.

The formulations of the therapeutic agent include those suitable forparenteral as well as non-parenteral administration. Exemplaryadministration modalities include oral, buccal, topical, nasal,subcutaneous, intramuscular, and intravenous, among others. Formulationssuitable for parenteral administration are preferred.

The formulations comprising the therapeutic agent of the presentinvention may conveniently be presented in unit dosage forms and may beprepared by any of the methods well known in the art of pharmacy. Suchmethods generally include the step of bringing the therapeutic agentsinto association with a carrier which constitutes one or more accessoryingredients. Typically, the formulations are prepared by uniformly andintimately bringing the therapeutic agent into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into dosage forms of the desiredformulation.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the therapeutic agent, whichpreferably is isotonic with the blood of the recipient (e.g.,physiological saline solution). Such formulations may include suspendingagents and thickening agents or other microparticulate systems which aredesigned to target the therapeutic agent to the circulation or one ormore organs. The formulations may be presented in unit-dose ormulti-dose form.

In addition to the aforementioned ingredients, the formulations of thisinvention may further include one or more accessory ingredient(s)selected from diluents, buffers, flavoring agents, disintegrants,surface active agents, thickeners, lubricants, preservatives (includingantioxidants), and the like.

While one of skill in the art can determine the desirable dose in eachcase (including a unit dose for depot administration), a suitable doseof the therapeutic agent for achievement of therapeutic benefit, may,for example, be in a range of about 1 microgram (μg) to about 100milligrams (mg) per kilogram body weight of the recipient, or in a rangeof about 10 μg to about 50 mg per kilogram body weight, or in a range ofabout 10 μg to about 10 mg per kilogram body weight. The desired dosemay be presented as one dose or two or more sub-doses administered atappropriate intervals throughout the dosing period (e.g., one week, twoweeks, etc. . . . ). These sub-doses can be administered in unit dosageforms, for example, containing from about 10 μg to about 500 mg, or fromabout 50 μg to about 200 mg, or from about 50 μg to about 100 mg ofactive ingredient per unit dosage form. Alternatively, if the conditionof the recipient so requires, the doses may be administered as acontinuous infusion.

The mode of administration and dosage forms will of course affect thetherapeutic amount of the peptide active therapeutic agent that isdesirable and efficacious for a given treatment application. Forexample, orally administered dosages can be at least twice, e.g., 2-10times, the dosage levels used in parenteral administration methods.Depot formulations will also allow for significantly more therapeuticagent to be delivered, such that the agent will have a sustained releaseover time.

VIP that circulates in the plasma of normal individuals originates fromVIP-containing nerve fibers in the gastrointestinal tract and alsoreflects peptide overflow from vascular nerves (Cugini et al., 1991, RegPept 34: 141-8). Like most vasoactive proteins, VIP has a relativelyshort half-life. The half-life of VIP in blood is less than 2 minutes(Domschke et al., 1978, Gut 19: 1049-53; Burhol et al., 1978, Scand JGastroent 13: 807-813). One advantage of the modified VIPs describedherein is extended half-life or persistence in the body. In accordancewith certain embodiments of the invention, the VIP may be administeredfrom 1 to about 10 times per week, such as from 1 to about 5, or 1 toabout 3 times per week. The modified VIP or pharmaceutical compositioncomprising the same may be administered about once daily, or about everyother day, or about every third day, or about once a week.

In certain embodiments, the modified VIP is administered parenterally,such as by subcutaneous or intramuscular injection. The administrationmay be a unit dose of the modified VIP as described herein.

The modified VIP, when administered parenterally, may be administeredonce per day, or once or twice per week, or from once to five times permonth. In these embodiments, the modified VIP may be administered as asoluble fusion peptide, that persists in the circulation, as describedherein, to provide sustained activity with relatively infrequentadministration. The modified VIP may be administered as a drug depot, asalso described herein, to provide a sustained release of fusion peptideinto the circulation over time. See US 2007/0009602, which is herebyincorporated by reference.

Methods of Use

In other aspects, the invention provides methods of treating,ameliorating, or preventing a condition in a mammal. Such conditionsinclude a variety of cardiovascular, immunological (e.g., autoimmune),and neurological conditions. For example, the modified VIP may be usedto adjust the balance between pro-inflammatory and anti-inflammatoryeffectors in a patient, including a patient suffering from an autoimmunedisease or inflammatory condition. Exemplary indications for themodified VIP include hypertension, myocardial fibrosis, heart failure,cardiomyopathy, diabetes, chronic obstructive pulmonary disease (COPD),arthritis, inflammatory bowel disease (IBD), Parkinson's disease, braintrauma, and asthma, among others.

The invention thus provides a method for treating a variety ofconditions, including conditions characterized by autoimmunity orinflammation. The method comprises administering an effective amount ofthe modified VIP of the invention to a patient in need.

Hypertension

In various embodiments described herein, the present invention providesmethods of treating or preventing hypertension in a patient in need,comprising administering an effective amount of the modified VIP. Formsof hypertension treatable with the modified VIPs of the presentinvention include pulmonary hypertension, uncontrolled essentialhypertension, and resistant hypertension.

Pulmonary hypertension is a relatively rare but highly fatal diseasecharacterized by progressive pulmonary arterial hypertension andincreased thickening of smaller pulmonary arteries and arterioles,culminating in right ventricular (RV) failure (Said et al., 2007,Circulation 115: 1260-8). VIP has been linked to pulmonary and systemiccirculation. With respect to the pulmonary vascular bed and itsalterations in pulmonary hypertension, VIP relaxes pulmonary vascularsmooth muscle from several mammalian species in vitro, neutralizes orattenuates the actions of endothelin and other vasoconstrictors, reduceshypoxic pulmonary vasoconstriction, and inhibits the proliferation ofpulmonary vascular smooth muscle from patients with pulmonaryhypertension. Furthermore, VIP is a cotransmitter of the physiologicalnonadrenergic, noncholinergic system of pulmonary vascular smooth musclerelaxation. Moreover, VIP-containing nerves, normally plentiful in thepulmonary artery, have been reported to be absent in pulmonary arteriesfrom patients with pulmonary hypertension, and inhalation of the peptidehad a beneficial therapeutic effect on those patients (Petkov et al.,2003, J. Clin Invest. 111: 1339-1346). Finally, studies have shown thatVIP replacement therapy in VIP^(−/−) mice is capable of preventing or atleast slowing the progression of key pathological changes in pulmonaryhypertension (Said et al., 2007, Circulation 115: 1260-8). Thus,application of VIP to patients with pulmonary hypertension can beexpected to result in substantial improvement of hemodynamic andprognostic parameters of the disease (Petkov et al., 2003, J. ClinInvest. 111: 1339-1346).

Uncontrolled essential hypertension is blood pressure that isconsistently higher than normal when no cause for the high bloodpressure can be found. Essential hypertension is the most prevalenthypertension type, affecting 90-95% of hypertensive patients (Carreteroet al., 2000, Circulation 101: 329-35) and experts believe it is causedby several undiscovered factors. Concentrations of VIP are decreased instroke-prone, essential hypertensive rats (Mori et al., 1993, Jpn HeartJ. 34: 785-94) and use of human VIP with sterically stabilized liposomescan normalize systemic arterial pressure in spontaneously hypertensivehamsters (Onyuksel et al., 2006, Peptides 27: 2271-5).

Resistant hypertension is a form of high blood pressure that does notrespond to treatment (i.e. blood pressure remains high even when acombination of drugs is administered). The causes of poor blood pressurecontrol are numerous. The most likely causes are volume overload eitherdue to excess sodium intake, intolerance to medications, noncomplianceand secondary hypertension (Graves J W, 2000, Mayo Clin Prac 75:278-84). As a potent systemic vasodilator, VIP has utility for thetreatment and prevention of hypertension in patients producing thehallmarks of resistant hypertension.

Heart Disease

In additional embodiments, the present invention provides methods oftreating or preventing heart disease in a patient in need, comprisingadministering an effective amount of the modified VIP. Forms of heartdisease treatable with the modified VIPs of the present inventioninclude myocardial fibrosis, heart failure, and cardiomyopathy.

Changes in the synthesis and secretion of VIP in the heart appear toplay a role in the pathogenesis of several diseases, such as heartfailure and myocardial fibrosis (Dvoráková M C, 2005, Drug NewsPerspect. 18: 387-91). For instance, the concentration of VIP isdecreased significantly in both tissue from patients with cardiomyopathyand in cardiac tissue from animal models of heart failure (Unverferth etal., 1986, J. Lab Clin Med 108: 11-16). Furthermore, degradation of VIPis increased in hearts with fibrosis and consequently myocardial VIPconcentration decreases. Thus, decreased VIP appears to be an importantfactor in the pathogenesis of the disease (Ye et al., 2003, Acta PhysiolScand 179: 353-60) and decreased VIP concentrations are associated witha progressive worsening of heart failure. The use of the vasopeptidaseinhibitor omapatrilat, which is known to decrease the metabolicclearance rate of VIP, resulted in a decrease in systolic blood pressureas well as in a decrease in myocardial fibrosis when compared to control(Ye et al., 2004, Eur J Pharmacol 485: 235-42). A protective effect ofVIP was also reported in ischemic and repurfused myocardium (Kalfin etal., 1994, J Pharmacol Exp Ther 268: 952-8). Therefore, application ofthe modified VIPs of the present invention can be expected to have abeneficial effect in a variety of pathological conditions, includingheart failure, cardiomyopathy, and myocardial fibrosis.

Type 2 Diabetes Mellitus

In additional embodiments, the present invention provides methods oftreating or preventing diabetes in a patient in need, comprisingadministering an effective amount of the modified VIP. Specifically, themodified VIPs of the present invention have utility for the treatmentand prevention of type 2 diabetes mellitus.

Studies have shown that the VIP content of the gastric astrum andduodenum of diabetic rats is significantly lower than that of normalrats (Gozes et al., 2004, Best Pract Res Clin Endocrinol Metab 18:623-640). Low tissue levels of VIP in the gastroduodenal tract maycontribute in part to the abnormal gut motility observed in diabeticpatients (Adeghate et al., 2001, Arch Phys Bloc 109: 246-51). VIPstimulates insulin secretion from insulinoma cells, mouse pancreaticislets and perfused rat pancreas. Activation of VPAC1 has beenimplicated in elevating glucose output (Gozes et al., 2004, Best PractRes Clin Endocrinol Metab 18: 623-640), whereas the VIP receptor VPAC2is expressed in pancreatic islet β-cells and its activation causes anelevation of cyclic AMP and the stimulation of insulin secretion(DeSouza et al., 2009, Nature Reviews 8: 361-7). Furthermore, VIPstimulates glucagon secretion in humans, resulting in glucose releasefrom the liver. Taken together, these studies reveal that VIP hasextensive direct effects on glucose metabolism. Accordingly, VIP andmodified forms of VIP, such as the fusion peptides disclosed herein, maybe expected to be useful therapy for the treatment and prevention oftype 2 diabetes.

VPAC2 Receptor Preference

In some embodiments, such as where the modified VIP has a higherpreference for VPAC2 as compared to unmodified VIP, the modified VIP mayreduce inflammatory responses, such as delayed-type hypersensitivityresponses, in a patient. In some such embodiments, the modified VIPreduces the development of autoreactive T-cells. In these embodiments,the patient may have one or more conditions defined by TH1-typeinflammation or TH1 autoimmunity, such as arthritis (including RA),Inflammatory Bowel Disease (e.g., Crohn's Disease), type 1 diabetes,multiple sclerosis, transplantation rejection, Sjogren's syndrome,pancreatitis, uveoretinitis, keratitis, and septic shock.

VPAC1 Receptor Preference

In some embodiments, such as where the modified VIP has a higherpreference for VPAC1 as compared to unmodified VIP, the modified VIP maypromote TH1 inflammatory responses, such as delayed-typehypersensitivity responses, in a patients. In these embodiments, thepatient may have one or more conditions associated with TH2 immunity,such as asthma or chronic obstructive pulmonary disease (COPD).

COPD is a chronic inflammatory disease of the airways, which affects asmany as 8% of individuals in industrialized nations. There is anincrease in the number of woman and men suffering from COPD. Pulmonaryhypertension is a common symptom of chronic airflow obstruction, but theprecise mechanisms of increased vascular resistance are unclear.Potential causes of pulmonary hypertension in COPD include emphysematousdestruction of the capillary bed, remodeling of pulmonary vessels andhypoxic pulmonary vasoconstriction.

VIP is one of the most abundant molecules found in the respiratorytract. Due to its anti-inflammatory and bronchodilatory properties, ithas been proposed as a novel treatment for COPD and asthma. AlthoughVPAC1 up-regulation is dominant, both VPAC1 and VPAC2 are necessary foroptimal anti-inflammatory signaling (Burian et al., 2010, Peptides 31:603-8). Accordingly, treatment with VIP and modified forms of VIP, suchas the fusion peptides disclosed herein, may be expected to helpdecrease the chronic inflammation in the lung of COPD and asthmapatients.

The present invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference in their entirety for all purposes.

EXAMPLES Example 1 Cloning of VIP-ELP Constructs

The DNA sequence for the VIP peptide was as described in Simoncsits etal. (Synthesis, cloning and expression in Escherichia coli of artificialgenes coding for biologically active elongated precursors of thevasoactive intestinal polypeptide, Eur. J. Biochem. 1988,178(2):343-350, which is herein incorporated by reference in itsentirety for all purposes), except that residue 17 was the nativemethionine and did not have either of the described C-terminalextensions (See SEQ ID NO. 16).

Two initial variants were made, one with a methionine at the N-terminus,due to the required ATG start codon, (PB1046, SEQ ID NO. 17) and onewith the tripeptide MAA at the N-terminus (PB1047, SEQ ID 18). Themethionine on PB1046 would normally be removed by methionineaminopeptidase (MA) but as histidine is the second residue and one ofthe least favored amino acids at this position for MA, the methionine isnot removed. The methionine on PB1047 was removed to leave AA, which canthen be removed in vitro or in vivo by DPPIV to give the histidine asthe N-terminal residue. The VIP DNA sequence was cloned into vectorpPB1031 (see FIG. 3) carrying the ELP1-120 DNA sequence to give anexpression cassette under the control of the T7 promoter.

The synthetic oligonucleotides P0045 (SEQ ID NO. 31), P0048 (SEQ ID NO.32), P0064 (SEQ ID NO. 33) and P0065 (SEQ ID NO. 34) were annealedtogether, digested with the restriction enzyme XbaI and ligated into theplasmid pPB1031 which had been digested with the restriction enzymesXbaI/KpnI to give expression plasmid pPB1046 (see FIG. 4).

The synthetic oligonucleotides P0066 (SEQ ID NO. 35), P0064 (SEQ ID NO.33), P0067 (SEQ ID NO. 36) and P0065 (SEQ ID NO. 34) were annealedtogether, digested with the restriction enzyme XbaI and ligated into theplasmid pPB1031 which had been digested with the restriction enzymesXbaI/KpnI to give expression plasmid pPB1047 (see FIG. 5).

In addition, and assuming that the N-terminus was not an absoluterequirement for activity, a C-terminal fusion was also made, pPB1048(see FIG. 6). The synthetic oligonucleotides P0068 (SEQ ID NO. 37) andP0069 (SEQ ID NO. 38) were annealed together and ligated into theplasmid pPB1031 which had been digested with the restriction enzymesBglI/NheI to give the expression plasmid pPB1048.

Example 2 Expression of VIP-ELP Constructs

The E. coli production strain BLR (Novogen) was transformed with theplasmids pB1046, pPB1047 and pPB1048 and grown in rich medium in shakeflasks at 37° C. overnight. The cell pellets were resuspended in TE pH8.0 buffer, lysed through a microfluidizer (Microfluidics), centrifugedto remove the insoluble material and the product purified from theresulting soluble lysate by ‘transitioning’ (ref) with the addition ofNaCl to 3M. The samples were taken through a further two rounds oftransitioning to give the final purified samples. These were analyzed bySDS-PAGE and PB1046 and PB1047 were found to give two bands (see FIG.7). Assuming this was as a result of proteolysis the cultures were grownagain but this time, before lysis, were heated to 60° C. of 15 minutes.Analysis by 10% Tris-Acetate NuPAGE gel indicted that the proteolysishad been inhibited (see FIG. 7).

The proteolysis was, most likely, within the peptide and probably closeto the junction of peptide and ELP as no breakdown was seen on theC-terminal fusion PB1048. That proteolysis could be prevented by heatdenaturing of protease(s) before lysis of the cells, which would tend toimplicate a periplasmic protease(s) rather than a cytosolic protease, ora cytosolic protease that was activated or behaves differently uponlysis.

Example 3 Activity of Modified VIP-ELP Fusion Protein In Vitro

To measure the in vitro biological activity and potency of VIP orVIP-ELP fusion proteins, a cell-based bioassay was used. The assaymeasures the increase in intracellular cyclic adenosine monophosphate(cAMP) concentration in response to treatment with VIP or VIP-ELP fusionproteins in Chinese Hamster Ovary (CHO) cells that have been engineeredto express either the human Vasoactive Intestinal Peptide Receptor 2(VPAC2) or the human Vasoactive Intestinal Peptide Receptor 1 (VPAC1).Both VIP and VIP-ELP fusion proteins can stimulate production of cAMP inthese cells, indicating that the fusion proteins retain the ability tobind and activate the receptor. Since the amount of cAMP accumulation incells after receptor-mediated ligand binding and activation is directlyproportional to the amount of intact peptide or fusion protein present,the assay can be used to determine bioactivity and relative potency.

In this example, the activity of VIP-ELP fusion proteins PB1046 andPB1047 was tested. Construct PB1046 contains VIP with a Met at theN-terminus and construct PB1047 contains VIP with Ala-Ala at itsN-terminus. Both constructs have ELP(1-120) at their C-terminus. In thefirst experiment, the activity of the constructs was tested using CHOcells expressing the VIP receptor VPAC2. After 30 minute incubations ofvarious concentrations of the fusion proteins with the cell, the cellswere lysed and the amount of cAMP produced was measured using acommercial kit. PB1047 was DPP-IV treated prior to the addition to thecells. FIG. 8 shows the result. As shown, modified VIP fusion proteinPB1046 is somewhat more active than native VIP protein, while PB1047 isless active.

The activity of PB1046 and PB1047 was also tested using CHO cellsexpressing the VIP receptor VPAC1. After 30 minute incubations ofvarious concentrations of the fusion proteins with CHO cells, cells werelysed and the amount of cAMP produced was measured using a commercialkit. PB1047 was DPP-IV treated prior to the addition to the cells. FIG.9 shows the result. This time, modified VIP fusion protein PB1046 ismuch less active than native VIP protein, while the relative activity ofPB1047 against native VIP is about the same as it was in the test forVPAC2 receptor. These results suggest that PB1046 selectively activatesVPAC2 receptor over VPAC1 receptor.

Example 4 Blood Pressure Effect of VIP-ELP Fusion Protein

The activity of the modified VIP-ELP fusion protein PB1047 was alsotested in vivo. Specifically, effects of VIP-ELP fusion protein on bloodpressure were tested. Spontaneously hypertensive rats were treatedsubcutaneous with PB1047 (10 mg/kg) or buffer control and their bloodpressures were measured at several points after administration of thefusion protein. Five animals were used for each group and the graphsshow the average and the standard deviation. PB1047 significantlyreduced systolic and diastolic blood pressure in these animals for atleast 12 hours post administration (see FIG. 10), indicating that theVIP-ELP fusion protein is active, and can be potentially used aspharmaceuticals in treating VIP-related diseases.

Example 5 Additional VIP-ELP Fusion Proteins

DPP IV treatment of PB1047 resulted in removal of both AA and HS fromthe N-terminus, and inactivation of the peptide. Plasmid pPB1064 wastherefore constructed, where the N-terminus was changed to MAAHG, SEQ IDNO: 45, instead of MAAHS, SEQ ID NO: 46, because HG is more resistant toDPP IV than HS.

Plasmid pPB1056 was also constructed, which encodes a VIP with anoppositely charged linker (SEQ ID NO. 20) based on the VPGXG, SEQ ID NO:3, repeat before ELP.

Example 6 Cloning, Expression, and Analysis of an Additional VIP-ELPFusion Protein, PB1120

The VIP DNA sequence was cloned into vector pPB1120 (SEQ ID NO: 48) (seeFIG. 11) carrying the ELP1-120 DNA sequence to give an expressioncassette under the control of the T7 promoter. Next, the E. coliproduction strain BLR was transformed with the pPB1120 plasmid and grownin rich medium as described above. Samples of the resulting VIP-ELP1-120fusion peptide, PB1120, were purified and analyzed via SDS-PAGE.

The activity of the PB1120 fusion peptide was tested in vitro. Theactivity was tested using an assay utilizing CHO cells expressing VIPreceptor (VPAC1) as described above in Example 3. As FIG. 12demonstrates, PB1120 was approximately 1.4 fold less active than thenative VIP peptide on the VPAC1 receptor. By comparison, the constructPB1046 which contains an N-terminal methionine residue was approximately11-fold less active than the native VIP peptide. Over the course ofmultiple experiments, PB1120 was anywhere from 1.4- to 6-fold lessactive than the native VIP peptide on the VPAC1 receptor.

FIG. 13 illustrates the activity of PB1120 for the VPAC2 receptor. Likethe results seen for the VPAC1 receptor, PB1120 show slightly lessactivity (˜1.5 fold less) than the native VIP peptide for VPAC2.However, in contrast to the results seen with VPAC1, PB1046 wasequipotent for VPAC2 as compared to the native peptide. Over the courseof multiple experiments, PB1120 was anywhere from 1.5- to 7-fold lessactive than the native VIP peptide on the VPAC2 receptor.

Example 7 Pharmacokinetic Profile of Modified VIP-ELP Fusion ProteinPB1120

In addition to the biological potency assays described above, thepharmacokinetic profile of the VIP-ELP fusion protein PB1120 was alsoexamined. Monkeys were given single subcutaneous (SC) injections (dosedat 3 mg/kg) of PB1120 and plasma drug concentrations were measured dailyover the course of one week. Three animals were used and the graphs showthe average and the standard deviation. More than half of the initialdose of PB1120 remained in the circulation to day 4 (see FIGS. 14A and14B, which illustrate the mean plasma concentrations of PB1120 after SCadministration using linear and semi-logarithmic axes, respectively).

Based upon this data, there appears to be a prolonged absorption phaseafter subcutaneous administration of PB1120, consistent with slowabsorption from the site of administration. The apparent eliminationhalf-life (t %), based on the decay of plasma concentrations, rangedfrom 9.9 to 45.8 h and likely reflects the slow absorption rather thantrue elimination. These data indicate that the VIP-ELP fusion proteinhas a dramatically extended half-life in comparison to native VIP andcan potentially be administered at extended intervals (e.g. may beadministered about once daily, about every other day, about every thirdday, or about once weekly).

Example 8 Effects of Modified VIP-ELP Fusion Protein PB1120 on BloodPressure

To measure the effects of the modified VIP-ELP fusion protein PB1120 onsystolic, diastolic, and mean arterial blood pressure, rats were givensingle, subcutaneous injections of 0.1 mg/kg, 1 mg/kg, or 5 mg/kg ofPB1120 and evaluated over 3-hr intervals. FIGS. 15A, 15B, and 15C showthe average change in systolic, diastolic, and mean arterial pressure,respectively. FIG. 15D shows the average heart rate over 3 hr intervalsfollowing administration of PB1120. As FIGS. 15A-C demonstrate, ratsinjected with either 1 mg/kg or 5 mg/kg of PB1120 showed significantreductions in systolic, diastolic, and mean arterial pressure 9 hrspost-injection, indicating that VIP-ELP fusion protein PB1120 canpotentially be administered for the purpose of treating or preventinghypertension in afflicted individuals.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

1. A method of treating hypertension, comprising administering to asubject a pharmaceutical composition comprising a Vasoactive IntestinalPeptide Receptor (VPAC) agonist comprising the amino acid sequence ofSEQ ID NO:13 having an N-terminal methionine, and an elastin-likepolypeptide (ELP) at the C-terminus, and one or more pharmaceuticallyacceptable excipients, wherein the ELP comprises at least 90 repeatunits of VPGXG (SEQ ID NO: 3), where X is independently selected fromVal, Ala, and Gly.
 2. The method of claim 1, wherein the VPAC agonisthas increased binding preference for VPAC2 versus VPAC1.
 3. The methodof claim 1, wherein the ELP prolongs the absorption phase from aninjection site and extends the circulatory half-life of the peptideagonist.
 4. The method of claim 1, wherein X is Val, Ala, and Gly in aratio of 5:2:3.
 5. The method of claim 1, wherein the pharmaceuticalcomposition is administered parenterally.
 6. The method of claim 5,wherein the pharmaceutical composition is administered subcutaneously,intramuscularly, or intravenously.
 7. The method of claim 1, wherein theELP comprises at least 120 repeat units of VPGXG (SEQ ID NO: 3), where Xis independently selected from Val, Ala, and Gly.
 8. The method of claim7, wherein X is Val, Ala, and Gly in a ratio of 5:2:3.
 9. The method ofclaim 1, wherein the VPAC agonist comprises the amino acid sequence ofSEQ ID NO:14.
 10. The method of claim 1, wherein the hypertension isselected from the group consisting of including pulmonary hypertension,uncontrolled essential hypertension, resistant hypertension, andpulmonary arterial hypertension.
 11. A method of treating hypertension,comprising administering to a subject a pharmaceutical compositioncomprising a Vasoactive Intestinal Peptide Receptor (VPAC) agonistcomprising the amino acid sequence of SEQ ID NO:13 having an N-terminalmethionine, and an elastin-like polypeptide (ELP) at the C-terminus, andone or more pharmaceutically acceptable excipients, wherein the ELPcomprises at least 90 units of VPGXG (SEQ ID NO:3), where X is Val, Ala,and Gly at a ratio of 5:2:3.
 12. The method of claim 11, wherein theVPAC agonist has increased binding preference for VPAC2 versus VPAC1.13. The method of claim 11 wherein the ELP prolongs the absorption phasefrom an injection site and extends the circulatory half-life of thepeptide agonist.
 14. The method of claim 11, wherein theelastin-like-polypeptide comprises 120 units of VPGXG (SEQ ID NO: 3).15. The method of claim 11, wherein the pharmaceutical composition isadministered parenterally.
 16. The method of claim 15, wherein thepharmaceutical composition is administered subcutaneously,intramuscularly, or intravenously.
 17. The method of claim 11, whereinthe VPAC agonist comprises the amino acid sequence of SEQ ID NO:14. 18.The method of claim 11, wherein the hypertension is selected from thegroup consisting of including pulmonary hypertension, uncontrolledessential hypertension, resistant hypertension, and pulmonary arterialhypertension.